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(CDCl3, 400.15 MHz): d=1.33 (t, 12H, J=7.2 Hz; 4CH2CH3); 3.63
(q, 8H, J=7.2 Hz; 4CH2CH3), 4.73 (d, 2H, J=6.0 Hz; NHCH2), 5.13
(s, 2H; 3’’-CH2C6H5), 5.18 (s, 2H; 2’’-CH2C6H5), 6.81 (d, 2H, J=2.3 Hz;
H-4, H-5), 6.89 (dd, 2H, J=9.6 and J=2.3 Hz; H-2, H-7), 6.92 (dd,
1H, J=8.0 and J=1.2 Hz; H-4’’), 7.01 (dd, 1H, J=8.0 and J=
7.6 Hz; H-5’’), 7.15 (d, 1H, J=7.6 Hz; H-6’’), 7.30–7.40 and 7.46–7.48
(2 m, 14H; H-Ar), 8.20 (d, 2H, J=8.0 Hz; H-3’, H-5’), 8.40 ppm (t,
1H, J=6.0 Hz; NHCH2); 13C NMR (CDCl3, 100.62 MHz): d=12.8
(CH2CH3), 39.5 (NHCH2), 46.3 (CH2CH3), 71.2 and 75.0 (CH2C6H5), 96.6
(C-4, C-5), 113.3 (C-4’’), 114.4 (C-2, C-7), 122.0 (C-6’’), 124.5 (C-5’’),
127.7, 128.1 (C-3’, C-5’), 128.5, 128.6, 128.7, 128.8, 129.6, 132.3,
138.0, 142.5 (C-2’’), 151.9 (C-3’’), 155.7, 158.1, 166.0 ppm (CONH);
MS (ESI): m/z: 744 [M+].
chelate iron ions. The coordination geometry of the iron center
provided by both ligands was elucidated by mass-spectromet-
ric analysis and semiempirical calculations, which revealed that
a “salicylate-type” coordination to the FeIII center takes place.
The optical properties reported herein show that the
RosCat1 ligand exhibited more interesting fluorescent proper-
ties than RosCat2. Therefore, the fluorescence response of
RosCat1 to the presence of FeIII ions in aqueous buffer at
physiological pH values showed concentration-dependent
quenching of the fluorescence intensity.
Our results have shown that the use of an inverted amide
function with an additional methylene unit is a suitable syn-
thetic solution for the conjugation of fluorescent moieties with
the catechol receptor, thus preserving fluorescent properties
that are absent when the most common amide configuration
is used. We believe that RosCat1 presents itself as a promising
prototype molecule for the design of FeIII probes that take
advantage of the high affinity and selectivity provided by its
catechol unit.
Synthesis of conjugate 5: A mixture of rosamine 3 (0.12 mmol,
0.05 g), dimethoxybenzoic acid (0.24 mmol, 0.04 g), EDC
(0.25 mmol, 0.04 g), HOBt (0.25 mmol, 0.04 g), and DIPEA
(0.24 mmol, 0.04 mL) in dry DMF (0.8 mL) was placed in a reaction
vial (10 mL), which was closed in a N2 atmosphere and placed in
the cavity of a CEM microwave reactor. The reaction vial was irradi-
ated (1 min up to 758C and 20 min at 758C at a power maximum
of 100 W). The resulting mixture was purified by flash chromatog-
raphy with CHCl3/methanol (9:1) as the eluent to afford conjugate
1
5 in 45% yield (31.6 mg). H NMR (CDCl3, 400.15 MHz): d=1.36 (t,
Experimental Section
12H, J=7.2 Hz; 4CH2CH3); 3.69 (q, 8H, J=7.2 Hz; 4CH2CH3),
3.97 (s, 3H; OCH3), 4.10 (s, 3H; OCH3), 6.86 (d, 2H, J=2.4 Hz; H-4,
H-5), 6.98 (dd, 2H, J=9.6 and J=2.4 Hz; H-2, H-7), 7.16 (dd, 1H,
J=8.4 and J=1.6 Hz; H-4’’), 7.26 (dd, 1H, J=8.4 and J=7.8 Hz; H-
5’’), 7.43 (d, 1H, J=8.4 Hz; H-3’, H-5’), 7.49 (d, 2H, J=9.6 Hz; H-1,
H-8), 7.82 (dd, 1H, J=7.8 and J=1.6 Hz; H-6’’), 8.17 (d, 2H, J=
8.4 Hz; H-2’, H-6’), 10.45 ppm (s, 1H; NH); 13C NMR (CDCl3,
100.62 MHz): d=13.6 (CH2CH3), 47.1 (CH2CH3), 57.1 and 62.8 (2
OCH3), 97.5 (C-4, C-5), 114.2 (C-1a, C-8a), 115.1 (C-2, C-7), 117.1 (C-
4’’), 121.3 (C-3’, C-5’), 123.8 (C-6’’), 125.8 (C-5’’), 127.3 (C-1’), 127.9,
131.6 (C-2’, C-6’), 133.1 (C-1,8), 141.6 (C-4’), 148.3 (C-2’’), 153.6 (C-
3’’), 156.4 (C-3,6), 158.1 (C-9), 158.9 (C-4a, C-5a), 164.6 ppm (CONH).
Reagents and solvents were purchased as reagent grade and used
without further purification, unless otherwise stated. NMR spectra
were recorded on a Bruker Avance III 400 spectrometer operated
at 400.15 and 100.62 MHz for the H and 13C nuclei and equipped
1
with pulse-gradient units capable of producing magnetic-field
pulsed gradients in the z direction of 50.0 GcmÀ1. For ligand
RosCat2, the NMR spectra were recorded on a Bruker Avance III
HD 600 spectrometer operated at 600.13 and 150.92 MHz for the
1H and 13C nuclei and equipped with pulse-gradient units capable
of producing magnetic-field pulsed gradients in the z direction of
1
6.57 GcmÀ1. Two-dimensional H/1H correlation spectra (COSY), gra-
dient-selected 1H/13C heteronuclear single quantum coherence
Synthesis of RosCat1: A solution of boron trichloride in dichloro-
methane (1m, 2 mL) was dropped slowly into an ice-bath-cooled
suspension of 4 (0.10 g, 0.14 mmol) in dry dichloromethane (8 mL)
in a N2 atmosphere. The mixture was stirred at room temperature
for 18 h. Methanol (18 mL) was added to stop the reaction. After
removal of the solvent under vacuum, the residue was precipitated
with methanol/acetone to afford RosCat1 as a deep-violet solid
1
(HSQC), and H/13C heteronuclear multiple-bond coherence (HMBC)
spectra were acquired by using standard Bruker software. Mass
spectra were acquired by Unidade De Espectrometria De Masas
(Santiago de Compostela, Spain) and microanalyses were acquired
by Unidad De Anµlisis Elemental (Santiago de Compostela, Spain).
Flash chromatography was carried out on silica gel Merck (230–
400 mesh). Electronic absorption spectra were recorded on
a Varian Cary bio50 spectrophotometer thermostabilized at 25.08C,
and fluorescence measurements were performed on a Varian Cary
Elipse spectrofluorometer equipped with a constant-temperature
multicell cell holder (25.0Æ0.18C), with 5 mm slits width for excita-
tion and emission. Mass spectra were acquired on a Bruker Ultra-
1
(56.7 mg, 68%). H NMR ([D6]DMSO, 400.15 MHz): d=1.22 (t, 12H,
J=7.0 Hz; 4CH2CH3); 3.67 (q, 8H, J=7.0 Hz; 4CH2CH3), 4.50 (d,
2H, J=6.0 Hz; NHCH2), 6.62 (t, 1H, J=7.6 Hz; H-5’’), 6.68 (d, 1H,
J=7.6 Hz; H-4’’), 6.72 (dd, 1H, J=7.6 and J=2.0 Hz; H-6’’), 7.00 (d,
2H, J=2.3 Hz; H-4, H-5), 7.14 (dd, 2H, J=9.6 and J=2.3 Hz; H-2,
H-7), 7.24 (d, 2H, J=9.6 Hz; H-1, H-8), 7.64 (d, 2H, J=8.4 Hz; H-2’,
H-6’), 8.20 (d, 2H, J=8.4 Hz; H-3’, H-5’), 8.72 (brs, 1H; NH), 9.19–
9.21 ppm (m, 2H; OH); 13C NMR ([D6]DMSO, 100.62 MHz): d=12.5
(CH2CH3), NHCH2 under the DMSO signal, 45.4 (CH2CH3), 96.1, 112.6,
114.3, 114.6, 118.7, 125.9, 127.7, 129.6, 131.5, 134.6, 135.5, 142.9,
145.2, 155.2, 155.7, 157.4, 165.8 ppm (CONH); 1H NMR (CD3OD,
400.15 MHz): d=1.31 (t, 12H, J=7.0 Hz; 4CH2CH3); 3.69 (q, 8H,
J=7.0 Hz; 4CH2CH3), 4.60 (s, 2H; NHCH2), 6.67 (dd, 1H, J=8.0
and J=7.6 Hz; H-5’’), 6.75 (dd, 1H, J=8.0 and J=1.6 Hz; H-4’’),
6.78 (dd, 1H, J=7.6 and J=1.6 Hz; H-6’’), 6.98 (d, 2H, J=2.4 Hz; H-
4, H-5), 7.07 (dd, 2H, J=9.4 and J=2.4 Hz; H-2, H-7), 7.32 (d, 2H,
J=9.4 Hz; H-1, H-8), 7.57 (d, 2H, J=8.0 Hz; H-2’, H-6’), 8.13 ppm (d,
2H, J=8.0 Hz; H-3’, H-5’); 13C NMR (CD3OD, 100.62 MHz): d=11.8
(CH2CH3), 39.6 (NHCH2), 45.9 (CH2CH3), 96.5 (C-4, C-5), 113.3 (C-1a,
C-8a), 114.6 (C-2, C-7), 114.7 (C-4’’), 119.7 (C-5’’), 120.5 (C-6’’), 125.3
(C-1’’), 128.0 (C-3’, C-5’), 130.0 (C-2’, C-6’), 131.8 (C-1, C-8), 135.8 (C-
fleXtreme MALDI-TOF/TOF equipped with
(200 Hz; Bruker Daltonik GmbH, Bremen, Germany).
a smartbeam laser
Synthesis
Synthesis of conjugate 4: A mixture of rosamine 1 (0.05 mmol,
20.7 mg), 2,3-dibenzyloxybenzylamine[8] (0.09 mmol, 28.7 mg), EDC
(0.05 mmol, 8.1 mg), HOBt (0.05 mmol, 7.1 mg), and DIPEA
(0.11 mmol, 0.02 mL) in dry DMF (0.3 mL) was placed in a reaction
vial (10 mL), which was closed in a nitrogen atmosphere and
placed in the cavity of a CEM microwave reactor. The reaction vial
was irradiated (1 min up to 758C and 20 min at 758C at a power
maximum of 100 W). The resulting mixture was purified by flash
chromatography with CHCl3 and then CHCl3/methanol (9:1) as the
1
eluents. Conjugate 4 was obtained in 77% yield (27.9 mg). H NMR
Chem. Eur. J. 2015, 21, 15692 – 15704
15701
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